The disclosure relates to the preparation and purification of polysaccharide-coated supports. More particularly, the disclosure relates to methods and kits to remove non-covalently bound polysaccharide polymer from polysaccharide-coated particles while at the same time reducing the size, changing shape and/or enhancing permeability of such polymer.
In the fields of medicine and clinical chemistry, many studies and determinations of physiologically reactive species such as cells, proteins, enzymes, cofactors, nucleic acids, substrates, antigens and antibodies, for example, are carried out using conjugates involving assay molecules such as, for example, specific binding pair (sbp) members or members of a signal-producing system (sps), e.g., labels, conjugated to supports. Various assay techniques that involve the binding of sbp members are known. These assay techniques generally also involve an sps member, e.g., a label, used in the detection part of the assay.
Polysaccharides, particularly dextran and dextran derivatives, have been conjugated to supports such as, for example, particles, to increase the hydrophilicity of the surface of the support and to provide conjugation sites for covalent attachment of molecules such as, for example, sbp members and sps members, to the surface of the support. The resulting conjugates of sbp members and supports permit specific binding of a complementary sbp member to the surface of the support with greatly reduced non-specific binding.
The support may be covalently coupled to the polysaccharide coat or layer by reaction between functional groups of the support and functional groups of the polysaccharide thereby resulting in the covalent attachment of the polysaccharide to the support. In some instances the functional groups of the support are amine reactive functional groups and the functional groups of the polysaccharide are amine functional groups. In some instances the functional groups of the support are amine groups and the functional groups of the polysaccharide are amine reactive functional groups. The amine reactive functional group may be, for example, an aldehyde group or a carboxyl group.
In addition to the covalent attachment of the polysaccharide to the support, there is also a certain amount of polysaccharide that becomes non-covalently bound to the support. After the covalent attachment of the polysaccharide to the support, but prior to attachment of an assay molecule, this non-covalently bound polysaccharide should be removed. Many processes have been employed in the past to remove non-covalently bound polysaccharide from a support. Typically, such processes involve extensively washing particles with various buffers and surfactants. In some instances sonication has also been used in the treatment of such supports. In another approach repeated washings followed by centrifugation have been utilized. Washing and other procedures for removing non-covalently bound polysaccharide are very slow and labor intensive and have been found to be less than adequate in reducing the amount of non-covalently bound polysaccharide to acceptable or negligible levels. The difficulties of such removal become more imposing as the scale of the preparation of the polysaccharide-bound supports increases such as in, for example, scale-up in manufacturing.
There is, therefore, a need to develop a procedure for reducing to a negligible level the amount of non-covalently bound polysaccharide in the preparation of polysaccharide-bound supports.